Research in Dr. Yu's laboratory spans two areas. The first is the safety of plasma and plasma-derived products, specifically, freedom from contamination with well-known viruses (such as hepatitis C virus or hepatitis A virus) or emerging pathogenic agents (such as TT virus, parvovirus B19, and others). The second is the purity and potency of immunoglobulins and other therapeutic products derived from plasma of either human or animal origin. Current projects focus on (1) developing specific nucleic acid test (NAT) methods to detect, quantify, and characterize the pathogens; (2) evaluating inactivation and/or removal procedures for these pathogens; (3) performing follow-up studies to evaluate FDA MedWatch reports of possible viral transmissions by plasma products; (4) detecting and characterizing complexing and/or neutralizing antibodies against the pathogens utilizing either in-vitro or animal models; (5) establishing the purity (or impurity) of therapeutic plasma products and studying the relationship between molecular integrity and potency; (6) formulating NAT (e.g., HCV RNA, HAV RNA and B19 DNA) and potency (e.g., anti-RhoD) standards or panels for the FDA and/or World Health Organization (WHO); and (7) conducting or participating in collaborative studies for NAT or potency working standards/International Standards sponsored by the FDA and/or WHO. The research goals are to ensure that therapeutic products prepared from plasma are safe and effective, and to establish a body of reliable information necessary for the development and evaluation of new, safe, and efficacious products. Viral Safety of Plasma Derived Products. Both hepatitis A virus (HAV) and parvovirus B19 are non-enveloped viruses and difficult to be inactivated. Some fractionators have voluntarily used minipool screening of plasma by NAT methods to limit viral levels of both HAV RNA and B19 DNA in manufacturing pools. We participated in providing one of the candidate materials and in testing them in multi-laboratory collaborative studies leading to the establishment of first International Standard (IS) for corresponding B19 DNA and HAV RNA for NAT assays. As a result, the respective CBER working standard has also been established and consensus levels assigned (106 copies/mL B19 DNA; 104 copies/mL HAV RNA). The WHO IS (106 IU/mL upon reconstitution) for B19 DNA was established in late 2000 while that for HAV RNA (105 IU/mL when reconstituted) was established in Feb 2003. We continued to monitor levels of B19 DNA contamination in plasma-derived products. The effect of minipool NAT screening voluntarily implemented by manufacturers on the B19 DNA levels is in progress. In collaboration with the CDC, we investigated whether a reported MedWatch case of B19 was causally related to two implicated lots of human plasma derived factor VIII concentrates (AHF) made by a manufacturer. Our data suggest strongly that the reported MedWatch case of B19 transmission was causally related to the infusion of the implicated lot of AHF and that the case could have prevented if the B19 minipool screening of plasma implemented. Manuscript is in preparation. We have been collaborating with Drs. Harvey Alter and Naomi Luban in the study entitled "Transfusion-Related Infections Prospectively Studied" (abbreviated as "TRIPS" study). The study enrolls blood donors and prospectively follows blood recipients in order to establish ongoing surveillance of the incidence of breakthrough infections from known transfusion-transmitted agents and establish a repository of donor and recipient samples so that any newly emerging infectious agent can be rapidly evaluate for its threat to the blood supply. We are performing tests for B19 DNA and B19 antibodies on the blood specimens provided. The study has begun and is in progress. We also collaborated with Dr. Kevin Brown of NIH and Dr. Gerold Zerlauth of Baxter BioScience to assess B19 infectivity of those sequentially collected plasma donation samples from a plasmapheresis donor by utilizing an in-vitro cell culture system. Our preliminary data indicate that the presence of 104 geq/mL of B19 DNA in the absence of any anti-B19 antibody was infectious whereas once anti-B19 IgG was present in plasma, even the presence of 106 geq/mL of B19 DNA was not infectious. Functional and Chemical Evaluation of Immunoglobulin and Albumin Products. The apparent viral safety of plasma derivatives with respect to parvovirus B19 and HAV has been attributed to the presence of appreciable anti-B19 and anti-HAV IgG antibodies in the starting plasma pools and hence the resulting products. We continued to monitor the levels of these two antibodies in immunoglobulin products. The monitoring is needed to ensure that the minipool NAT screening of plasma for either B19 DNA or HAV RNA recently implemented voluntarily by some manufacturers has not adversely compromise the product safety. In collaboration with scientists in OVRR, we continued to monitor levels of anti-measles antibodies in IGIV products. Varying neutralizing antibody levels were found among IGIV products. Studies have been in progress to determine whether there is a trend of decreasing levels of anti-measles in products in recent years and to evaluate the neutralizing capabilities in relation to the presence of IgG fragments and subclasses. In collaboration with Dr. S Thorpe of NIBSC, a candidate global standard for the potency assay of anti-D was formulated and characterized from the two anti-D immunoglobulin products made in the US, one kindly donated by Bayer Corp and the other acquired by CBER from Ortho-clinical Dignostics, Inc. The candidate standard was calibrated against the International Reference Preparation (IRP) for anti-D immunoglobulin along with two reserve candidate reference preparations developed in Europe and an existing CBER potency standard, Lot 3, in an international collaborative study jointly sponsored by WHO/NIBSC, CBER, and European Pharmacopoeia (Eur Ph). Our laboratory utilizing the newly developed flow cytometry method was also one of 25 laboratories participated in testing. The global (WHO/CBER/Eur Ph) standard has finally been established in 2003 and has an assigned anti-D potency of 285 IU/ampoule. Recently, two batches of IGIV made in UK associated with hemolytic events were found to have high levels of anti-D by Dr. S Thorpe et al in NIBSC. They proposed a titer of 8 as the limit for batch acceptance and release by Eur Ph. To control assay variability and sensitivity, these authors developed a simple, direct, microtiter plate-based hemagglutination (HA) assay method and formulated a candidate IGIV reference preparation containing ca. 0.05 IU of anti-D per mL. An international collaborative study sponsored by NIBSC/Eur Ph was carried out to check out the suitability of the new method and the candidate reference preparation in order to set the maximal anti-D titer in IGIV products. We set up the new method and were one of the participating laboratories in testing. As a result of the study, the level of anti-D in IGIV distributed in Europe will be specified. We subsequently assayed many IGIV lots made within the last 3 years. The positive reference preparation had a titer of 8. The majority of the IGIV lots had no detectable anti-D. However, several lots had a titer of 8, and one lot had a titer of 16. Because varying amounts of IGIV are used clinically, depending on the indication, it is difficult to set a limit of anti-D in the product that would avoid all hemolytic adverse events. Nevertheless, to avoid compromising the safety of IGIV products, steps such as plasma screening and final product testing may need to be taken to minimize the levels of anti-D. We continued to monitor molecular integrity by HPLC for conformance lots of IGIV and Albumin products submitted by manufacturers for licensure. In addition, some experimental or licensed specific immunoglobulin preparations, which include Botulinum Immune Globulins derived either equine or human origin, were tested. The objectives of the latter analysis were to determine whether antibodies against botulinum toxin retain their molecular integrity and hence their antibody activities during storage for special counter-terrorism needs. This project incorporates FY2002 projects 1Z01BQ004020-08 and 1Z01BQ004021-08.